Real-Time PCR (qPCR) | Easy Animated Biology

Two samples reach the same height at the end of a PCR… does that mean they started with the same amount of DNA? In this video, we follow the light cycle by cycle to understand what a qPCR curve tells us, and how to use it to estimate how much DNA was there at the start. Real-time PCR (qPCR), from tubes to curves: fluorescence, the threshold, Cq, efficiency, and how to turn all of it into a number. In this video, you'll learn: How the instrument turns fluorescence into an amplification curve Dyes and hydrolysis (TaqMan) probes: how they produce the signal, and how the melting curve helps you read it What the threshold, Cq and the logarithmic scale mean How starting DNA amount and amplification efficiency are related, and what perfect doubling (2ⁿ) means Absolute quantification with a standard curve, and relative quantification to compare the expression of a gene Why the "RT" in RT-PCR stands for reverse transcription Technical and biological replicates, the no-template control, and a first look at digital PCR New to PCR? Watch the basics first: PCR — The Basics, on this channel. Chapters 00:00 From PCR to real-time PCR 01:03 Watching the reaction with light 01:56 Dyes and the melting curve 03:17 Hydrolysis (TaqMan) probes 04:41 The amplification curve and Cq 06:08 Cq, starting amount and efficiency 07:44 Absolute quantification: the standard curve 09:29 Relative quantification 10:03 Controls, digital PCR and wrap-up The animations are teaching diagrams. The curves are illustrative models, and the treatment follow-up curve is an illustrative trend: it does not represent patient data. References cited on screen Russell Higuchi et al. (1993). Kinetic PCR Analysis: Real-time Monitoring of DNA Amplification Reactions. Nature Biotechnology. https://doi.org/10.1038/nbt0993-1026 Stephen A. Bustin et al. (2009). The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry. https://doi.org/10.1373/clinchem.2008... Further reading C. A. Heid et al. (1996). Real time quantitative PCR. Genome Research. https://doi.org/10.1101/gr.6.10.986 Credits Narration: generated locally from a synthetic version of the creator's own voice, reviewed for this video. Music: original, composed for this channel. Sound effects: Pixabay. Animation: original. Did you like it? Subscribe to Make Things Viral, leave a like, and tell me in the comments which topic you want next. If you found a mistake or something wasn't clear, I read those too. Learn, and become your best version. #qPCR #PCR #MolecularBiology